Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi
Inventors
Emalfarb, Mark A. • Punt, Peter J. • van Zeijl, Cornelia • van den Hondel, Cornelius • Verdoes, Jan Cornelis • Burlingame, Richard P.
Assignees
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Publication Number
US-9862956-B2
Publication Date
2018-01-09
Expiration Date
Abstract
The invention is generally directed to modified filamentous fungal host cells comprising one or more nucleic acids encoding one or more polypeptides under the control of one or more promoters that are functional in said cells. Methods of using the modified cells to express one or more polypeptides are also disclosed, including methods of screening cells transformed with one or more expression vectors comprising nucleic acids derived from synthetic or genomic nucleic acids including, cDNAs. Methods of purifying one or more polypeptides or complexes comprising one or more polypeptides expressed in the modified cells, intended for use as substrates in structure/function studies, as therapeutic agents, as diagnostic reagents, or as human or animal vaccines, are also disclosed.
Core Innovation
The invention relates to methods of producing one or more polypeptides from a modified fungal cell. Each nucleic acid includes at least one coding region encoding at least one polypeptide operably-linked to at least one regulatory region comprising at least one promoter active in the cell, and the polypeptides or a complex comprising the polypeptides are purified from the cells or from cell-free medium obtained from a culture.
The modified fungal cell is selected from Chrysosporium strain UV18-25 deposited as VKMF-3631 D, Trichoderma longibrachiatum strain X-252, or a derivative or mutant of either. The cell has a deletion of at least one protease gene selected from alp1, alp2, and pep4, and has a culture viscosity phenotype in which culture viscosity is less than 200 cP at the end of fermentation under suspension culture with adequate nutrients and optimal or near-optimal conditions.
The invention further provides screening and isolation approaches that use transferable reproductive elements formed in suspension. A plurality of polypeptides encoded by a combinatorial library of vectors are expressed after formation of transferable reproductive elements, separation into a liquid or solid medium for monoclonal culture or monoclonal colonies, and subsequent culturing, and the presence or amount of polypeptides or a complex comprising the polypeptides is identified or measured from cells or cell-free medium.
Claims Coverage
The document includes four independent claims. Across these claims, the inventive features center on modified fungal cells with protease gene deletions and a culture viscosity phenotype, the expression and purification of polypeptides from promoter-active nucleic acids, and use of transferable reproductive elements with monoclonal cultures or monoclonal colonies and identification or measurement of polypeptides or complexes.
Production of polypeptides using modified protease-deficient fungal cells with low culture viscosity
A method of producing one or more polypeptides from a modified fungal cell comprising one or more nucleic acids, wherein each nucleic acid comprises at least one coding region encoding at least one polypeptide operably-linked to at least one regulatory region comprising at least one promoter active in said cell, the method comprising culturing the cell under conditions conducive to expression and purifying the one or more polypeptides or a complex comprising the one or more polypeptides from the cells or from cell-free medium, wherein the modified fungal cell is a fungal cell selected from Chrysosporium strain UV18-25 deposited as VKMF-3631 D, Trichoderma longibrachiatum strain X-252, or a derivative or mutant, has a deletion of at least one protease gene selected from alp1, alp2, and pep4, and has a culture viscosity phenotype wherein culture viscosity is less than 200 cP at the end of fermentation.
Monoclonal generation from transferable reproductive elements for polypeptide production
A method of producing one or more polypeptides from a modified fungal cell comprising one or more nucleic acids, wherein each nucleic acid comprises at least one coding region encoding at least one polypeptide operably-linked to at least one regulatory region comprising at least one promoter active in said cell, the method comprising culturing the cell under conditions conducive to formation of a plurality of transferable reproductive elements in suspension, separating the suspension of transferable reproductive elements into a liquid or solid medium suitable for the growth of monoclonal cultures or monoclonal colonies, culturing the monoclonal cultures or monoclonal colonies under conditions conducive to expression of the polypeptides encoded by the nucleic acids, and purifying the one or more polypeptides or a complex comprising the polypeptides from the cells or from cell-free medium, wherein the modified fungal cell is a fungal cell selected from Chrysosporium strain UV18-25 deposited as VKMF-3631 D, Trichoderma longibrachiatum strain X-252, or a derivative or mutant, has a deletion of at least one protease gene selected from alp1, alp2 and pep4, and has a culture viscosity phenotype wherein culture viscosity is less than 200 cP at the end of fermentation.
Screening polypeptides from a combinatorial vector library using transferable reproductive elements in modified fungal cells
A method of screening a plurality of polypeptides encoded by a combinatorial library of vectors for a functional activity or a structural property of interest in a modified fungal cell, wherein the modified fungal cell comprises one or more nucleic acids with coding regions encoding the polypeptides operably-linked to regulatory regions comprising promoters active in said cell, the method comprising culturing the cell under conditions conducive to formation of a plurality of transferable reproductive elements in suspension, separating the suspension into a liquid or solid medium suitable for the growth of monoclonal cultures or monoclonal colonies, culturing the monoclonal cultures or monoclonal colonies under conditions conducive to expression of the polypeptides, and identifying the presence or measuring the amount of the polypeptides or a complex comprising the polypeptides obtained from the cells or from cell-free medium, wherein the modified fungal cell is a fungal cell selected from Chrysosporium strain UV18-25 deposited as VKMF-3631 D, Trichoderma longibrachiatum strain X-252, or a derivative or mutant, has a deletion of at least one protease gene selected from alp1, alp2 and pep4, and has a culture viscosity phenotype wherein culture viscosity is less than 200 cP at the end of fermentation.
Specific UV18#100f derivative fungal cells with defined gene deletions
The fungal cell used in the method is a derivative of strain UV18#100f and is selected from the group consisting of UV18#100f Δalp1, UV18#100f Δpyr5 Δalp1, UV18#100.f Δalp1 Δpep4 Δalp2, UV18#100.f Δpyr5 Δalp1 Δpep4 Δalp2, and UV18#100.f Δpyr4 Δpyr5 Δalp1 Δpep4 Δalp2.
Overall, the claim set defines modified fungal cells characterized by deletions of protease genes and a culture viscosity phenotype, and applies these cells to producing and purifying polypeptides expressed from promoter-active nucleic acids and to screening a plurality of polypeptides from a combinatorial vector library using transferable reproductive elements with monoclonal cultures or monoclonal colonies.
Stated Advantages
Reduced entangled morphology.
Uniform/stable integration.
Improved transformation frequency.
Compatibility with automated robotic HTS formats.
Higher secreted protein/biomass ratios.
Suitability for producing and assaying proteins and complexes from clonal fungal cultures.
Documented Applications
Producing one or more polypeptides from a modified fungal cell, including purifying polypeptides or complexes from cells or cell-free medium obtained from the culture.
Screening a plurality of polypeptides encoded by a combinatorial library of vectors for a functional activity or a structural property of interest in a modified fungal cell, including identifying or measuring polypeptides or complexes from cells or cell-free medium.
Screening using high-throughput screening step (d) and identifying or measuring via one selected from immunoassay, detection, analytical, and cell-based techniques, including Western blot and mass spectrometry.
Producing, expressing, and assaying heterogeneous or heteromultimeric proteins including immunoglobulins and receptors from clonal fungal cultures.
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